CYP4C16 gene and application thereof in regulating and controlling cyhalothrin resistance of periplaneta americana

Abstract

The invention discloses the technical field of biology, and particularly relates to a CYP4C16 gene and application of the CYP4C16 gene in regulation and control of resistance of periplaneta americana to cyhalothrin, and the nucleotide sequence of the CYP4C16 gene is shown as SEQ ID NO.1.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to... CYP4C1_6 Genes and their application in regulating American cockroach resistance to cyhalothrin. Background Technology

[0002] American cockroaches are common pests that can transmit many diseases to humans in daily life by carrying pathogens, bacteria, and parasite eggs, such as Enterobacteriaceae. Enterobacterium ), Pseudomonas spp. ( Pseudomonas Bacteria such as [list of bacteria] can adversely affect multiple systems in humans, including the urinary, digestive, and respiratory systems. During the koji-making process, certain pests specifically harm the koji. The American cockroach is one such pest. Due to their large population size, the koji that is eaten is riddled with holes, sometimes leaving only an empty shell. This weakens the aroma of the koji, severely damages the microbial community, and leads to a significant decrease in the saccharification and liquefaction capabilities of the koji, potentially resulting in reduced yield.

[0003] Chemical pesticides remain the most common and effective pest management method for controlling American cockroaches. Commonly used agents include lambda-cyhalothrin, chlorpyrifos, imidacloprid, and triazophos, sprayed on the cockroach's habitat and areas of activity. This method offers advantages such as high killing efficiency, low cost, and ease of use. However, long-term use of chemical pesticides pollutes the environment, leads to pesticide resistance in American cockroaches, and reduces the effectiveness of pesticides. Therefore, proactively implementing resistance management strategies before widespread resistance to lambda-cyhalothrin develops in American cockroaches is of great significance for the sustainable use of lambda-cyhalothrin. Summary of the Invention

[0004] The present invention aims to provide a method for regulating the resistance of American cockroaches to cyhalothrin. CYP4C1_6 Gene.

[0005] In this plan CYP4C1_6 Gene, CYP4C1_6 The nucleic acid sequence of the gene is shown in SEQ ID NO.1.

[0006] SEQIDNO.1: .

[0007] Research has found that, CYP4C1_6 The gene is involved in the detoxification process of the American cockroach against the highly effective cyhalothrin; therefore, the present invention also claims protection for the following: CYP4C1_6 Application of genes in regulating resistance of American cockroaches to cyhalothrin.

[0008] CYP4C1_6The expression levels of the gene in different insect stages were: adults > larvae; the expression levels in different sexes were: female adults > male adults.

[0009] The larger the American cockroach, the stronger its ability to regulate resistance to cyhalothrin.

[0010] Knockout CYP4C1_6 Gene knockout (using technologies such as CRISPR / Cas9) can reduce the resistance of American cockroaches to cyhalothrin. By knocking out this gene (reducing its function), the detoxification ability of American cockroaches to cyhalothrin can be directly weakened, making them sensitive to the insecticide again.

[0011] The encoding possesses the above CYP4C1_6 The protein of a gene or its active fragment. CYP4C1_6 Proteins and their active fragments, as functional entities, can be used to determine their detoxification efficiency against cyhalothrin (such as metabolic rate, substrate specificity, etc.) through in vitro enzyme activity assays, revealing the molecular mechanism of resistance. At the same time, as drug screening targets, they can be used for high-throughput screening of compounds that can inhibit its activity (such as small molecule inhibitors).

[0012] contain CYP4C1_6 Recombinant vectors or host cells are used to reconstitute genes. Recombinant vectors (such as plasmids and viral vectors) enable stable expression of the gene in a heterologous host (such as E. coli or insect cells), while the host cell serves as a "biofactory" for large-scale in vitro production. CYP4C1_6 Proteins that meet experimental needs such as enzyme activity detection, structural analysis, and inhibitor screening.

[0013] Targeted silencing CYP4C1_6 dsRNA, siRNA, or expression vectors. dsRNA and siRNA can be specifically inhibited using RNA interference technology. CYP4C1_6 Gene transcription or translation reduces gene expression levels; expression vectors can continuously generate silencing molecules in vivo, prolonging the silencing effect.

[0014] CYP4C1_6 Inhibitors. Inhibitors can directly bind to... CYP4C1_6 The protein inhibits its detoxification activity (such as preventing it from metabolizing cyhalothrin), thus preventing resistant American cockroaches from degrading the insecticide and restoring its killing effect.

[0015] Expression or knockout CYP4C1_6 Transgenic insect cells, model organisms, or microorganisms expressing this gene can be used to simulate a high-resistance state and study its detoxification mechanism; models with the gene knocked out can simulate a low-resistance state and allow for comparative analysis of resistance-related phenotypic changes. Attached Figure Description

[0016] Figure 1 For each stage of the American cockroachCYP4C1_6 Gene expression levels.

[0017] Figure 2 for CYP4C1_6 Transmembrane domain prediction diagram.

[0018] Figure 3 for CYP4C1_6 Screenshot of protein domain prediction.

[0019] Figure 4 American cockroach CYP4C1_6 Predicted protein secondary structure diagram.

[0020] Figure 5 American cockroach CYP4C1_6 Predicted tertiary structure diagram of protein.

[0021] Figure 6 This is a prediction graph of the signal peptide.

[0022] Figure 7 American cockroach CYP4C1_6 Predicted protein phosphorylation sites.

[0023] Figure 8 American cockroach CYP4C1_6 Genetic phylogenetic evolutionary tree. Detailed Implementation

[0024] The following detailed description illustrates the specific implementation method: 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Experimental Subjects The American cockroaches used in this experiment were all purchased from an American cockroach farm in Guangxi (the raw material supplier for Kangfuxin Liquid of Sichuan Good Doctor Pharmaceutical Group), and were an indoor artificially bred population. They were placed in eggshell containers with breathable mesh, and kept in a dark environment at a room temperature of 25-30℃. They were fed white rice every 24 hours, and the American cockroach incubators were cleaned regularly.

[0025] 1.1.2 Instruments, Equipment and Related Reagents Table 1-1 Instruments and reagents involved in the experiment

[0026] 1.2 Experimental Methods 1.2.1 Sample Pretreatment American cockroaches were divided into four groups: male adults (M), female adults (F), older nymphs (H), and younger larvae (L). Ten cockroaches were collected from each group. Lambda-cyhalothrin was selected as the pesticide for stress. The two groups of adults were treated with a sublethal concentration of 857.434 mg / L, while the older and younger larvae were treated with a sublethal concentration of 332.802 mg / L. Treatment lasted for 1 hour. The four groups of cockroaches were then anesthetized using carbon dioxide gas. After removal, the heads, wings, and legs of the cockroaches were removed using scissors and forceps. The cockroaches were quickly placed into 2 mL EP tubes and temporarily stored in liquid nitrogen, then transferred to a -80°C freezer for later use.

[0027] 1.2.2 RNA Extraction Table 1-2 RNA Extraction Kits

[0028] According to the instructions for the RNA extraction kit, and referring to the steps for animal tissue samples: 1. Take three American cockroach samples from each group, stored at -80℃, and place them in a mortar pre-cooled with liquid nitrogen. Grind continuously with a pestle until the sample becomes a white powdery substance. During grinding, liquid nitrogen should be added frequently to maintain the low temperature of the sample. Take 100 mg of sample powder into a 1.5 mL EP tube, add 1 mL of TransZol UP and 0.2 mL of RNA Extraction Agent, and then vortex for at least five minutes to ensure that the sample mixture is thoroughly mixed. Centrifuge the resulting centrifuge tube at 10000×4℃ for 15 minutes. At this time, the mixture in the centrifuge tube will separate into an upper layer (colorless aqueous phase), a middle layer (cockroach tissue), and a lower layer (pink organic phase). Aspirate 400 μL of the aqueous phase and transfer it to a new EP tube. Add 400 μL of anhydrous ethanol, mix gently, transfer to a centrifuge column, and centrifuge at 12000×4℃ for 1 minute. Remove the centrifuge column and discard the eluent.

[0029] 2. Add 500 μL CCB9 to the centrifuge column, incubate at room temperature for 12000 × 1 minute, and discard the effluent.

[0030] 3. Repeat the above steps once.

[0031] 4. Add 500 mL of WB9 (prepared with anhydrous ethanol), centrifuge at 12000 rpm for 30 seconds at room temperature, and discard the effluent.

[0032] 5. Repeat the above step once.

[0033] 6. Centrifuge at 12000× for 2 minutes at room temperature to remove residual ethanol.

[0034] 7. Place the centrifuge column in an RNase-free tube, add 30 μL of RNase-free water, let stand for one minute, elute at 12000×1 minute, and retain the eluent.

[0035] 8. Extract 1 μL of RNA from each sample to measure the OD value, perform gel electrophoresis on 500-1000 ng RNA to determine the quality of RNA extraction, and store the obtained RNA samples at -80℃.

[0036] 1.2.3 cDNA Synthesis Prepare a 20 μL reverse transcription system: Table 1-3 Reverse Transcription System

[0037] Prepare four 0.2 mL centrifuge tubes and add the following mixture: 500-1000 ng RNA (each added to a different centrifuge tube), then add 1 μL Random primer, 1 μL Enzyme Mix, gDNARemover, 6 μL RNase-free Water, and 10 μL ES Reaction Mix.

[0038] Reaction conditions: Incubate at 42°C for 15 minutes; heat at 85°C for 5 seconds to inactivate EasyScript® RT / RI and gDNARemover; store at 4°C.

[0039] 1.2.4 RT-qPCR amplification Table 1-4 Forward and Reverse Primer Sequences

[0040] Table 1-5 RT-qPCR reaction system

[0041] Vortex the prepared RT-qPCR system for a few seconds, then centrifuge with a handheld centrifuge to ensure the system is well mixed and settles to the bottom.

[0042] Set the reaction conditions: Table 1-6 qPCR reaction conditions

[0043] 2 -△△Ct Analytical method: 1. Ct value: In quantitative real-time PCR, the Ct value is the number of cycles in which the detected fluorescence signal exceeds the background noise threshold. The smaller the Ct value, the higher the initial amount of the target gene.

[0044] 2. ΔCt value: The ΔCt value is a calculation of relative expression level, representing the Ct value of the target gene minus the Ct value of the reference gene. The reference gene is usually a gene that is stably expressed in different samples.

[0045] 3. ΔΔCt value: The ΔΔCt value is calculated to compare the differences in gene expression levels between different samples. It represents the ΔCt value of the target sample minus the ΔCt value of the reference sample. The smaller the ΔΔCt value, the lower the expression level of the target gene in the target sample.

[0046] 4.2 -△△Ct Value, 2 -△△Ct The value is a calculation that converts the ΔΔCt value into a relative expression level. It represents the multiple of the relative expression level of the target sample relative to the relative expression level of the reference sample. If 2 -△△Ct A value of 1 indicates that the expression levels of the target sample and the reference sample are equal; if 2... -△△Ct A value greater than 1 indicates that the expression level of the target sample is higher than that of the reference sample; if 2... -△△Ct A value less than 1 indicates that the expression level of the target sample is lower than that of the reference sample.

[0047] PCR amplification product amount = starting template amount × (1+e)^Ct; since e is assumed to be 1 here; Then the amount of PCR amplification product = the amount of initial template × 2^Ct; Then the amount of initial template = the amount of PCR amplification product / (2^Ct). That is, the amount of initial template = the amount of PCR amplification product × 2^(-Ct).

[0048] The Ct values ​​differ when the PCR amplification product amounts reach the same level because the initial template amounts differ. When the PCR amplification product amounts of the sample and the internal reference gene are the same, a division calculation is needed to calculate the ratio difference of their corresponding initial template amounts. Given: Test gene template amount = PCR amplification product amount × 2^(-Ct test); Internal reference gene template amount = PCR amplification product amount × 2^(-Ct internal reference); then, R = Test gene template amount / Internal reference gene template amount = PCR amplification product amount × 2^(-Ct test) / PCR amplification product amount × 2^(-Ct internal reference); therefore, R = 2^((-Ct test) - (-Ct internal reference)) = 2^(-ΔCt). Here, the R value is simply the ratio difference between each test sample and its corresponding internal reference gene. Dividing the 2^(-ΔCt) of all samples and the control group obtained above by the 2^(-ΔCt) of the control group yields 2^(-ΔΔCt).

[0049] 1.2.5 American cockroach CYP4C1_6 Gene bioinformatics analysis Using ExpasyProtparamTool CYP4C1_6 Physicochemical properties analysis of gene proteins; Using Interpro CYP4C1_6 Gene analysis and protein structure prediction; Use the Wolfpsort online tool to... CYP4C1_6 Subcellular localization prediction of gene amino acid sequences; Use the SOPMA online tool to... CYP4C1_6 Secondary structure analysis of the gene's protein sequence; Predict using the SWISS-MODEL online tool CYP4C1_6 The tertiary structure of proteins in genes; Use the Signalp tool to CYP4C1_6 Predicting signal peptides and cleavage sites for gene proteins; Using NetPhosS tools CYP4C1_6 Predict phosphorylation sites; Using MEGA11 software, CYP4C1_6 Genes and their similar gene inputs were constructed using PhylogeneticAnalysis. CYP4C1_6 Phylogenetic evolutionary tree of genes.

[0050] 2. Results and Discussion 2.1 Data Analysis The average Ct values ​​of the four groups of samples were obtained by RT-qPCR: 32.108 for young larvae, 33.162 for older larvae, 31.978 for male adults, and 32.206 for female adults. The data were analyzed using the 2-ΔΔCt method.

[0051] Through analysis CYP4C1_6 Gene expression trends at different insect stages and sexes are shown in the attached figure. Figure 1 As shown, from Figure 1 It was found that the expression level in the three insect stages was: adult > older larvae > younger larvae; and the expression level in females was slightly higher than that in males. Among the older larvae... CYP4C1_6 Gene expression levels are approximately twice that of young larvae; in female adults... CYP4C1_6 The gene expression level was twice that of older larvae and four times that of younger larvae; the expression level in male adults was 1.5 times that of older larvae and three times that of younger larvae; the expression level in female adults was slightly higher than that in male adults, approximately 1.3 times. Based on the body size of each group of samples: female adults > male adults > older larvae > younger larvae, the detoxification gene was inferred. CYP4C1_6Gene expression levels may be related to energy distribution and utilization rates within the American cockroach; larger American cockroaches exhibit stronger regulatory abilities against cyhalothrin. Based on this, we can boldly speculate on the detoxification genes of the American cockroach. CYP4C1_6 Expression levels are positively correlated with developmental stage. When using insecticides on American cockroaches, priority should be given to the larval stage rather than the adult stage. Control methods should also combine chemical, environmental, and biological approaches, with prevention as the primary focus.

[0052] 2.2 CYP4C1_6 Results of genetic bioinformatics analysis 2.2.1 CYP4C1_6 Gene nucleotide and amino acid sequence analysis American cockroaches were sequenced using the ORF Finder in NCBI. CYP4C1_6 Open reading frame (ORF) analysis of the gene's nucleotide sequence revealed that its ORF region is 936 bp long and encodes a total of 311 amino acids.

[0053] Table 2-1 Open Reading Frame Analysis

[0054] CYP4C1_6 The gene's amino acid sequence is as follows: MLRPWLKNDFLYSFTTKGKRNAECLRVLHGFTKRVIQERKSQLEGDFNRSPGTSTDEDAFVGKKKRKAFLDLLLESSIGDIKLTDEELREEVDTFMFEGHDTTSAGMCWALFLLGLHPDVQEKAYEEQKNIFQGSERS VTMKELNEMKYLERVIKETLRLYPSDFTTPAGCTVFFHIYNVHRNPEQFPNPDKFDPDNFLPERVAKRHPYAYIPFSAGPRNCIGQKFALLEEKTVLSYILRNYEVTSLDKRQDVNLMTELVLRPEKGINVSIKPRNK.

[0055] 2.2.2 Prediction of transmembrane domains Using TMHMM network tools CYP4C1_6 Protein transmembrane domain prediction, such as Figure 2 As shown: There are two transmembrane structures between 0 and 60.

[0056] 2.2.3 Physicochemical property analysis Using the Expasy Protparam Tool CYP4C1_6Physicochemical analysis of the protein revealed that it contains 41 negatively charged amino acid residues (Asp + Glu) and 42 positively charged amino acid residues (Arg + Lys), indicating that the amino acid sequence is positively charged. The chemical formula is C. 1441 H 2254 N 392 O 421 S 10 The gene's CDS region encodes 276 amino acids, with a relative molecular mass of 32126.72, an isoelectric point of 7.66 (slightly alkaline), an instability coefficient of 47.22 (greater than 40 indicates easy degradation), an adipose coefficient of 76.27, and an average hydrophobicity of -0.604, classifying it as a hydrophilic protein.

[0057] Table 2-2 Physicochemical Properties Analysis

[0058] 2.2.4 Protein Domain Prediction Using Interpro CYP4C1_6 Protein structure and prediction were performed, and the results are as follows: Figure 3 As shown: The representative structure of this protein is P450 type, which belongs to the Cytochrome P450 4 family of proteins. It has conserved sites of the Cytochrome P450 family. Homologous families show that this protein belongs to the Cyt-P450 superfamily.

[0059] 2.2.5 Subcellular localization prediction Use the Wolfpsort online tool to... CYP4C1_6 The amino acid sequence was used to predict subcellular localization, as follows: Table 2-3 Subcellular localization analysis

[0060] Subcellular localization analysis revealed that a total of 43 similar proteins were analyzed, showing... CYP4C1_6 Genes are relatively evenly distributed in the four locations of mitochondria, nucleus, cytoplasm, and plasma membrane, with a small amount distributed in other locations.

[0061] 2.2.6 Protein Spatial Structure Prediction Use the SOPMA online tool to... CYP4C1_6 Secondary structure analysis of protein sequences: Table 2-4 Protein Secondary Structure Prediction

[0062] The results are as follows Figure 4 As shown: CYP4C1_6The protein secondary structure analysis results showed that it consisted of 43.48% α-helices (120 in total), 5.07% β-sheets (14 in total), 39.13% random coils (108 in total), and 12.32% extended strands (34 in total). The structure was then predicted using the SWISS-MODEL online tool. CYP4C1_6 The tertiary structure of the protein, as shown below Figure 5 As shown, its secondary and tertiary structures are consistent, with α-helices and random coils as the main structures.

[0063] 2.2.7 Prediction of signal peptides and cleavage sites Signal peptides are short peptide chains that guide the translocation of proteins into the secretory pathway, promoting the secretory expression of collagen. Signal peptide prediction results are as follows... Figure 6 As shown: In CYP4C1_6 The protein amino acid sequence did not contain any signal peptide sites larger than the threshold of 0.45, indicating that no possible signal peptide sequences or signal peptide cleavage sites were found.

[0064] 2.2.8 Phosphorylation site prediction Using NetPhosS tools CYP4C1_6 Predict phosphorylation sites. Figure 7 As shown in the figure, S, T and Y represent serine, threonine and tyrosine, respectively. According to the prediction, a total of 43 sites have a prediction score higher than the threshold of 0.5, which are more likely phosphorylation sites.

[0065] 2.2.9 CYP4C1_6 Phylogenetic tree Access the NCBI Blast web tool and enter... CYP4C1_6 Gene amino acid sequences were selected, and nucleotide sequences of 10 similar genes were chosen. Using MEGA 11 software, the similar genes were input and constructed using Phylogenetic Analysis. CYP4C1_6 Phylogenetic evolutionary tree of genes, such as Figure 8 As shown.

[0066] This phylogenetic tree shows that the American cockroach... CYP4C1_6 Genes and Pacific cockroaches ( Diploptera punntata They are in the same branch, most closely related, and have the highest homology, followed by the German cockroach (…). Blattella germanica It is closely related to the brown planthopper ( ) and has a high degree of homology. nilaparvata lugens The phylogenetic relationship is the most distant, and the homology is the lowest.

[0067] 2.3 Prediction Tools Table 2-5 Forecasting Tools

[0068] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific structures and characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the structure of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. CYP4C1_6 Genes are characterized by: The nucleic acid sequence is shown in SEQ ID NO.

1.

2. The claim 1 CYP4C1_6 Application of genes in regulating resistance of American cockroaches to cyhalothrin.

3. As described in claim 2 CYP4C1_6 The application of genes in regulating resistance to cyhalothrin in American cockroaches is characterized by: CYP4C1_6 The expression levels of the gene in different insect stages were: adults > larvae; the expression levels in different sexes were: female adults > male adults.

4. As described in claim 2 CYP4C1_6 The application of genes in regulating resistance to cyhalothrin in American cockroaches is characterized by: The larger the American cockroach, the stronger its ability to regulate resistance to cyhalothrin.

5. The method according to claim 2 CYP4C1_6 The application of genes in regulating resistance to cyhalothrin in American cockroaches is characterized by: Knockout CYP4C1_6 Genes that reduce the resistance of American cockroaches to cyhalothrin.

6. The encoding possesses the features described in claim 1 CYP4C1_6 The protein of a gene or its active fragment.

7. Containing the contents of claim 1 CYP4C1_6 Gene recombination vectors or host cells.

8. Targeted silencing as described in claim 1 CYP4C1_6 The gene's dsRNA, siRNA, or expression vector.

9. The claim 1 CYP4C1_6 Gene inhibitors.

10. Expression or knockout of claim 1 CYP4C1_6 Genetically modified insect cells, model organisms, or microorganisms.

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